Cohesin-mediated Loop Research Articles

Interpreting the CTCF-mediated sequence grammar of genome folding with AkitaV2.

Interphase mammalian genomes are folded in 3D with complex locus-specific patterns that impact gene regulation. CTCF (CCCTC-binding factor) is a key architectural protein that binds specific DNA sites, halts cohesin-mediated loop extrusion, and enables long-range chromatin interactions. There are hundreds of thousands of annotated CTCF-binding sites in mammalian genomes; disruptions of some result in distinct phenotypes, while others have no visible effect. Despite their importance, the determinants of which CTCF sites are necessary for genome folding and gene regulation remain unclear. Here, we update and utilize Akita, a convolutional neural network model, to extract the sequence preferences and grammar of CTCF contributing to genome folding. Our analyses of individual CTCF sites reveal four predictions: (i) only a small fraction of genomic sites are impactful; (ii) impact is highly dependent on sequences flanking the core CTCF binding motif; (iii) core and flanking nucleotides contribute largely additively to the overall impact of a site; (iv) sites created as combinations of different core and flanking sequences have impacts proportional to the product of their average impacts, i.e. they are broadly compatible. Our analysis of collections of CTCF sites make two predictions for multi-motif grammar: (i) insulation strength depends on the number of CTCF sites within a cluster, and (ii) pattern formation is governed by the orientation and spacing of these sites, rather than any inherent specialization of the CTCF motifs themselves. In sum, we present a framework for using neural network models to probe the sequences instructing genome folding and provide a number of predictions to guide future experimental inquiries.

Mature chromatin packing domains persist after RAD21 depletion in 3D.

Understanding chromatin organization requires integrating measurements of genome connectivity and physical structure. It is well established that cohesin is essential for TAD and loop connectivity features in Hi-C, but the corresponding change in physical structure has not been studied using electron microscopy. Pairing chromatin scanning transmission electron tomography with multiomic analysis and single-molecule localization microscopy, we study the role of cohesin in regulating the conformationally defined chromatin nanoscopic packing domains. Our results indicate that packing domains are not physical manifestation of TADs. Using electron microscopy, we found that only 20% of packing domains are lost upon RAD21 depletion. The effect of RAD21 depletion is restricted to small, poorly packed (nascent) packing domains. In addition, we present evidence that cohesin-mediated loop extrusion generates nascent domains that undergo maturation through nucleosome posttranslational modifications. Our results demonstrate that a 3D genomic structure, composed of packing domains, is generated through cohesin activity and nucleosome modifications.

Permeable TAD boundaries and their impact on genome-associated functions.

TAD boundaries are genomic elements that separate biological processes in neighboring domains by blocking DNA loops that are formed through Cohesin-mediated loop extrusion. Most TAD boundaries consist of arrays of binding sites for the CTCF protein, whose interaction with the Cohesin complex blocks loop extrusion. TAD boundaries are not fully impermeable though and allow a limited amount of inter-TAD loop formation. Based on the reanalysis of Nano-C data, a multicontact Chromosome Conformation Capture assay, we propose a model whereby clustered CTCF binding sites promote the successive stalling of Cohesin and subsequent dissociation from the chromatin. A fraction of Cohesin nonetheless achieves boundary read-through. Due to a constant rate of Cohesin dissociation elsewhere in the genome, the maximum length of inter-TAD loops is restricted though. We speculate that the DNA-encoded organization of stalling sites regulates TAD boundary permeability and discuss implications for enhancer-promoter loop formation and other genomic processes.

YY1-controlled regulatory connectivity and transcription are influenced by the cell cycle.

Few transcription factors have been examined for their direct roles in physically connecting enhancers and promoters. Here acute degradation of Yin Yang 1 (YY1) in erythroid cells revealed its requirement for the maintenance of numerous enhancer-promoter loops, but not compartments or domains. Despite its reported ability to interact with cohesin, the formation of YY1-dependent enhancer-promoter loops does not involve stalling of cohesin-mediated loop extrusion. Integrating mitosis-to-G1-phase dynamics, we observed partial retention of YY1 on mitotic chromatin, predominantly at gene promoters, followed by rapid rebinding during mitotic exit, coinciding with enhancer-promoter loop establishment. YY1 degradation during the mitosis-to-G1-phase interval revealed a set of enhancer-promoter loops that require YY1 for establishment during G1-phase entry but not for maintenance in interphase, suggesting that cell cycle stage influences YY1's architectural function. Thus, as revealed here for YY1, chromatin architectural functions of transcription factors can vary in their interplay with CTCF and cohesin as well as by cell cycle stage.

Molecular basis for differential Igk versus Igh V(D)J joining mechanisms

In developing B cells, V(D)J recombination assembles exons encoding IgH and Igκ variable regions from hundreds of gene segments clustered across Igh and Igk loci. V, D and J gene segments are flanked by conserved recombination signal sequences (RSSs) that target RAG endonuclease1. RAG orchestrates Igh V(D)J recombination upon capturing a JH-RSS within the JH-RSS-based recombination centre1–3 (RC). JH-RSS orientation programmes RAG to scan upstream D- and VH-containing chromatin that is presented in a linear manner by cohesin-mediated loop extrusion4–7. During Igh scanning, RAG robustly utilizes only D-RSSs or VH-RSSs in convergent (deletional) orientation with JH-RSSs4–7. However, for Vκ-to-Jκ joining, RAG utilizes Vκ-RSSs from deletional- and inversional-oriented clusters8, inconsistent with linear scanning2. Here we characterize the Vκ-to-Jκ joining mechanism. Igk undergoes robust primary and secondary rearrangements9,10, which confounds scanning assays. We therefore engineered cells to undergo only primary Vκ-to-Jκ rearrangements and found that RAG scanning from the primary Jκ-RC terminates just 8 kb upstream within the CTCF-site-based Sis element11. Whereas Sis and the Jκ-RC barely interacted with the Vκ locus, the CTCF-site-based Cer element12 4 kb upstream of Sis interacted with various loop extrusion impediments across the locus. Similar to VH locus inversion7, DJH inversion abrogated VH-to-DJH joining; yet Vκ locus or Jκ inversion allowed robust Vκ-to-Jκ joining. Together, these experiments implicated loop extrusion in bringing Vκ segments near Cer for short-range diffusion-mediated capture by RC-based RAG. To identify key mechanistic elements for diffusional V(D)J recombination in Igk versus Igh, we assayed Vκ-to-JH and D-to-Jκ rearrangements in hybrid Igh–Igk loci generated by targeted chromosomal translocations, and pinpointed remarkably strong Vκ and Jκ RSSs. Indeed, RSS replacements in hybrid or normal Igk and Igh loci confirmed the ability of Igk-RSSs to promote robust diffusional joining compared with Igh-RSSs. We propose that Igk evolved strong RSSs to mediate diffusional Vκ-to-Jκ joining, whereas Igh evolved weaker RSSs requisite for modulating VH joining by RAG-scanning impediments.

Intergenic sequences harboring potential enhancer elements contribute to Axenfeld-Rieger syndrome by regulating PITX2.

Recent studies have uncovered that noncoding sequence variants may relate to Axenfeld-Rieger syndrome (ARS), a rare developmental anomaly with genetic heterogeneity. However, how these genomic regions are functionally and structurally associated with ARS is still unclear. In this study, we performed genome-wide linkage analysis and whole-genome sequencing in a Chinese family with ARS and identified a heterozygous deletion of about 570 kb (termed LOH-1) in the intergenic sequence between paired-like homeodomain transcription factor 2 (PITX2) and family with sequence similarity 241 member A. Knockout of LOH-1 homologous sequences caused ARS phenotypes in mice. RNA-Seq and real-time quantitative PCR revealed a significant reduction in Pitx2 gene expression in LOH-1-/- mice, while forkhead box C1 expression remained unchanged. ChIP-Seq and bioinformatics analysis identified a potential enhancer region (LOH-E1) within LOH-1. Deletion of LOH-E1 led to a substantial downregulation of the PITX2 gene. Mechanistically, we found a sequence (hg38 chr4:111,399,594-111,399,691) that is on LOH-E1 could regulate PITX2 by binding to RAD21, a critical component of the cohesin complex. Knockdown of RAD21 resulted in reduced PITX2 expression. Collectively, our findings indicate that a potential enhancer sequence that is within LOH-1 may regulate PITX2 expression remotely through cohesin-mediated loop domains, leading to ARS when absent.

YY1 Binding to Regulatory Elements That Lack Enhancer Activity Promotes Locus Folding and Gene Activation

Enhancers activate their cognate promoters over huge distances but how enhancer/promoter interactions become established is not completely understood. There is strong evidence that cohesin-mediated loop extrusion is involved but this does not appear to be a universal mechanism. Here, we identify an element within the mouse immunoglobulin lambda (Igλ) light chain locus, HSCλ1, that has characteristics of active regulatory elements but lacks intrinsic enhancer or promoter activity. Remarkably, knock-out of the YY1 binding site from HSCλ1 reduces Igλ transcription significantly and disrupts enhancer/promoter interactions, even though these elements are >10 kb from HSCλ1. Genome-wide analyses of mouse embryonic stem cells identified 2671 similar YY1-bound, putative genome organizing elements that lie within CTCF/cohesin loop boundaries but that lack intrinsic enhancer activity. We suggest that such elements play a fundamental role in locus folding and in facilitating enhancer/promoter interactions.

Multi-feature clustering of CTCF binding creates robustness for loop extrusion blocking and Topologically Associating Domain boundaries

Topologically Associating Domains (TADs) separate vertebrate genomes into insulated regulatory neighborhoods that focus genome-associated processes. TADs are formed by Cohesin-mediated loop extrusion, with many TAD boundaries consisting of clustered binding sites of the CTCF insulator protein. Here we determine how this clustering of CTCF binding contributes to the blocking of loop extrusion and the insulation between TADs. We identify enrichment of three features of CTCF binding at strong TAD boundaries, consisting of strongly bound and closely spaced CTCF binding peaks, with a further enrichment of DNA-binding motifs within these peaks. Using multi-contact Nano-C analysis in cells with normal and perturbed CTCF binding, we establish that individual CTCF binding sites contribute to the blocking of loop extrusion, but in an incomplete manner. When clustered, individual CTCF binding sites thus create a stepwise insulation between neighboring TADs. Based on these results, we propose a model whereby multiple instances of temporal loop extrusion blocking create strong insulation between TADs.

Lifting the curtain on loop extrusion barriers: Single-molecule insights into cohesin stalling

In this issue, Zhang etal.1 show that CTCF blocks cohesin-mediated loop extrusion in an orientation-dependent manner. Using single-molecule imaging assays, the authors find that dCas9 and R-loops can also stall extrusion.

Ultra-long-range interactions between active regulatory elements.

Contacts between enhancers and promoters are thought to relate to their ability to activate transcription. Investigating factors that contribute to such chromatin interactions is therefore important for understanding gene regulation. Here, we have determined contact frequencies between millions of pairs of cis-regulatory elements from chromosome conformation capture data sets and analyzed a collection of hundreds of DNA-binding factors for binding at regions of enriched contacts. This analysis revealed enriched contacts at sites bound by many factors associated with active transcription. We show that active regulatory elements, independent of cohesin and polycomb, interact with each other across distances of tens of megabases in vertebrate and invertebrate genomes and that interactions correlate and change with activity. However, these ultra-long-range interactions are not dependent on RNA polymerase II transcription or individual transcription cofactors. Using simulations, we show that a model of chromatin and multivalent binding factors can give rise to long-range interactions via bridging-induced clustering. We propose that long-range interactions between cis-regulatory elements are driven by at least three distinct processes: cohesin-mediated loop extrusion, polycomb contacts, and clustering of active regions.

CTCF is a DNA-tension-dependent barrier to cohesin-mediated loop extrusion

In eukaryotes, genomic DNA is extruded into loops by cohesin1. By restraining this process, the DNA-binding protein CCCTC-binding factor (CTCF) generates topologically associating domains (TADs)2,3 that have important roles in gene regulation and recombination during development and disease1,4–7. How CTCF establishes TAD boundaries and to what extent these are permeable to cohesin is unclear8. Here, to address these questions, we visualize interactions of single CTCF and cohesin molecules on DNA in vitro. We show that CTCF is sufficient to block diffusing cohesin, possibly reflecting how cohesive cohesin accumulates at TAD boundaries, and is also sufficient to block loop-extruding cohesin, reflecting how CTCF establishes TAD boundaries. CTCF functions asymmetrically, as predicted; however, CTCF is dependent on DNA tension. Moreover, CTCF regulates cohesin’s loop-extrusion activity by changing its direction and by inducing loop shrinkage. Our data indicate that CTCF is not, as previously assumed, simply a barrier to cohesin-mediated loop extrusion but is an active regulator of this process, whereby the permeability of TAD boundaries can be modulated by DNA tension. These results reveal mechanistic principles of how CTCF controls loop extrusion and genome architecture.

A de novo transcription-dependent TAD boundary underpins critical multiway interactions during antibody class switch recombination

Interactions between transcription and cohesin-mediated loop extrusion can influence 3D chromatin architecture. However, their relevance in biology is unclear. Here, we report a direct role for such interactions in the mechanism of antibody class switch recombination (CSR) at the murine immunoglobulin heavy chain locus (Igh). Using Tri-C to measure higher-order multiway interactions on single alleles, we find that the juxtaposition (synapsis) of transcriptionally active donor and acceptor Igh switch (S)sequences, an essential step in CSR, occurs via the interaction of loop extrusion complexes with a de novo topologically associating domain (TAD) boundary formed via transcriptional activity across S regions. Surprisingly, synapsis occurs predominantly in proximity to the 3' CTCF-binding element (3'CBE) rather than the Igh super-enhancer, suggesting a two-step mechanism whereby transcription of S regions is not topologically coupled to synapsis, as has been previously proposed. Altogether, these insights advance our understanding of how 3D chromatin architecture regulates CSR.

A cohesin traffic pattern genetically linked to gene regulation.

Cohesin-mediated loop extrusion has been shown to be blocked at specific cis-elements, including CTCF sites, producing patterns of loops and domain boundaries along chromosomes. Here we explore such cis-elements, and their role in gene regulation. We find that transcription termination sites of active genes form cohesin- and RNA polymerase II-dependent domain boundaries that do not accumulate cohesin. At these sites, cohesin is first stalled and then rapidly unloaded. Start sites of transcriptionally active genes form cohesin-bound boundaries, as shown before, but are cohesin-independent. Together with cohesin loading, possibly at enhancers, these sites create a pattern of cohesin traffic that guides enhancer-promoter interactions. Disrupting this traffic pattern, by removing CTCF, renders cells sensitive to knockout of genes involved in transcription initiation, such as the SAGA complexes, and RNA processing such DEAD/H-Box RNA helicases. Without CTCF, these factors are less efficiently recruited to active promoters.

Cohesin and CTCF complexes mediate contacts in chromatin loops depending on nucleosome positions

The spatial organization of the eukaryotic genome plays an important role in regulating transcriptional activity. In the nucleus, chromatin forms loops that assemble into fundamental units called topologically associating domains that facilitate or inhibit long-range contacts. These loops are formed and held together by the ring-shaped cohesin protein complex, and this can involve binding of CCCTC-binding factor (CTCF). High-resolution conformation capture experiments provide the frequency at which two DNA fragments physically associate in three-dimensional space. However, technical limitations of this approach, such as low throughput, low resolution, or noise in contact maps, make data interpretation and identification of chromatin intraloop contacts, e.g., between distal regulatory elements and their target genes, challenging. Herein, an existing coarse-grained model of chromatin at single-nucleosome resolution was extended by integrating potentials describing CTCF and cohesin. We performed replica-exchange Monte Carlo simulations with regularly spaced nucleosomes and experimentally determined nucleosome positions in the presence of cohesin-CTCF, as well as depleted systems as controls. In fully extruded loops caused by the presence of cohesin and CTCF, the number of contacts within the formed loops was increased. The number and types of these contacts were impacted by the nucleosome distribution and loop size. Microloops were observed within cohesin-mediated loops due to thermal fluctuations without additional influence of other factors, and the number, size, and shape of microloops were determined by nucleosome distribution and loop size. Nucleosome positions directly affect the spatial structure and contact probability within a loop, with presumed consequences for transcriptional activity.

Chromatin jets define the properties of cohesin-driven in vivo loop extrusion

Complex genomes show intricate organization in three-dimensional (3D) nuclear space. Current models posit that cohesin extrudes loops to form self-interacting domains delimited by the DNA binding protein CTCF. Here, we describe and quantitatively characterize cohesin-propelled, jet-like chromatin contacts as landmarks of loop extrusion in quiescent mammalian lymphocytes. Experimental observations and polymer simulations indicate that narrow origins of loop extrusion favor jet formation. Unless constrained by CTCF, jets propagate symmetrically for 1-2 Mb, providing an estimate for the range of invivo loop extrusion. Asymmetric CTCF binding deflects the angle of jet propagation as experimental evidence that cohesin-mediated loop extrusion can switch from bi- to unidirectional and is controlled independently in both directions. These data offer new insights into the physiological behavior of invivo cohesin-mediated loop extrusion and further our understanding of the principles that underlie genome organization.

Cohesin is required for long-range enhancer action at the Shh locus

The regulatory landscapes of developmental genes in mammals can be complex, with enhancers spread over many hundreds of kilobases. It has been suggested that three-dimensional genome organization, particularly topologically associating domains formed by cohesin-mediated loop extrusion, is important for enhancers to act over such large genomic distances. By coupling acute protein degradation with synthetic activation by targeted transcription factor recruitment, here we show that cohesin, but not CTCF, is required for activation of the target gene Shh by distant enhancers in mouse embryonic stem cells. Cohesin is not required for activation directly at the promoter or by an enhancer located closer to the Shh gene. Our findings support the hypothesis that chromatin compaction via cohesin-mediated loop extrusion allows for genes to be activated by enhancers that are located many hundreds of kilobases away in the linear genome and suggests that cohesin is dispensable for enhancers located more proximally.

You shall not pass! Unveiling the barriers for cohesin-mediated loop extrusion

Dequeker and colleagues performed elegant invivo, in silico, and invitro experiments to demonstrate that the MCM complex, an essential DNA replication factor, is an obstacle for the DNA loop formation by cohesin.

Cohesin-mediated loop anchors confine the locations of human replication origins

DNA replication occurs through an intricately regulated series of molecular events and is fundamental for genome stability1,2. At present, it is unknown how the locations of replication origins are determined in the human genome. Here we dissect the role of topologically associating domains (TADs)3–6, subTADs7 and loops8 in the positioning of replication initiation zones (IZs). We stratify TADs and subTADs by the presence of corner-dots indicative of loops and the orientation of CTCF motifs. We find that high-efficiency, early replicating IZs localize to boundaries between adjacent corner-dot TADs anchored by high-density arrays of divergently and convergently oriented CTCF motifs. By contrast, low-efficiency IZs localize to weaker dotless boundaries. Following ablation of cohesin-mediated loop extrusion during G1, high-efficiency IZs become diffuse and delocalized at boundaries with complex CTCF motif orientations. Moreover, G1 knockdown of the cohesin unloading factor WAPL results in gained long-range loops and narrowed localization of IZs at the same boundaries. Finally, targeted deletion or insertion of specific boundaries causes local replication timing shifts consistent with IZ loss or gain, respectively. Our data support a model in which cohesin-mediated loop extrusion and stalling at a subset of genetically encoded TAD and subTAD boundaries is an essential determinant of the locations of replication origins in human S phase.

MCM complexes are barriers that restrict cohesin-mediated loop extrusion

Eukaryotic genomes are compacted into loops and topologically associating domains (TADs)1–3, which contribute to transcription, recombination and genomic stability4,5. Cohesin extrudes DNA into loops that are thought to lengthen until CTCF boundaries are encountered6–12. Little is known about whether loop extrusion is impeded by DNA-bound machines. Here we show that the minichromosome maintenance (MCM) complex is a barrier that restricts loop extrusion in G1 phase. Single-nucleus Hi-C (high-resolution chromosome conformation capture) of mouse zygotes reveals that MCM loading reduces CTCF-anchored loops and decreases TAD boundary insulation, which suggests that loop extrusion is impeded before reaching CTCF. This effect extends to HCT116 cells, in which MCMs affect the number of CTCF-anchored loops and gene expression. Simulations suggest that MCMs are abundant, randomly positioned and partially permeable barriers. Single-molecule imaging shows that MCMs are physical barriers that frequently constrain cohesin translocation in vitro. Notably, chimeric yeast MCMs that contain a cohesin-interaction motif from human MCM3 induce cohesin pausing, indicating that MCMs are ‘active’ barriers with binding sites. These findings raise the possibility that cohesin can arrive by loop extrusion at MCMs, which determine the genomic sites at which sister chromatid cohesion is established. On the basis of in vivo, in silico and in vitro data, we conclude that distinct loop extrusion barriers shape the three-dimensional genome.

DNA Damage Response and Repair in Adaptive Immunity.

The diversification of B-cell receptor (BCR), as well as its secreted product, antibody, is a hallmark of adaptive immunity, which has more specific roles in fighting against pathogens. The antibody diversification is from recombination-activating gene (RAG)-initiated V(D)J recombination, activation-induced cytidine deaminase (AID)-initiated class switch recombination (CSR), and V(D)J exon somatic hypermutation (SHM). The proper repair of RAG- and AID-initiated DNA lesions and double-strand breaks (DSBs) is required for promoting antibody diversification, suppressing genomic instability, and oncogenic translocations. DNA damage response (DDR) factors and DSB end-joining factors are recruited to the RAG- and AID-initiated DNA lesions and DSBs to coordinately resolve them for generating productive recombination products during antibody diversification. Recently, cohesin-mediated loop extrusion is proposed to be the underlying mechanism of V(D)J recombination and CSR, which plays essential roles in promoting the orientation-biased deletional end-joining . Here, we will discuss the mechanism of DNA damage repair in antibody diversification.